Selective HDAC3 inhibitors

ABSTRACT

Provided herein are inhibitors of HDAC3, as well as methods of treatment comprising administering those compounds to a subject in need thereof.

PRIORITY BENEFIT

This application claims the benefit of U.S. Provisional Application Ser.No. 61/759,732, filed Feb. 1, 2013, the content of which is incorporatedherein in its entirety.

BACKGROUND OF THE INVENTION

A biological target of recent interest is histone deacetylase (HDAC)(see, for example, a discussion of the use of inhibitors of histonedeacetylases for the treatment of cancer: Marks et al. Nature ReviewsCancer 2001, 7,194; Johnstone et al. Nature Reviews Drug Discovery 2002,287). Post-translational modification of proteins through acetylationand deacetylation of lysine residues plays a critical role in regulatingtheir cellular functions. HDACs are zinc hydrolases that modulate geneexpression through deacetylation of the N-acetyl-lysine residues ofhistone proteins and other transcriptional regulators (Hassig et al.Curr. Opin. Chem. Biol. 1997, 1, 300-308). HDACs participate in cellularpathways that control cell shape and differentiation, and an HDACinhibitor has been shown effective in treating an otherwise recalcitrantcancer (Warrell et al. J. Natl. Cancer Inst. 1998, 90, 1621-1625).

At this time, eleven human HDACs, which use Zn as a cofactor, have beenidentified (Taunton et al. Science 1996, 272, 408-411; Yang et al. J.Biol. Chem. 1997, 272, 28001-28007; Grozinger et al. Proc. Natl. Acad.Sci. U.S.A. 1999, 96, 4868-4873; Kao et al. Genes Dev. 2000, 14, 55-66;Hu et al. J. Biol. Chem. 2000, 275, 15254-15264; Zhou et al. Proc. Natl.Acad. Sci U.S.A. 2001, 98, 10572-10577; Venter et al. Science 2001, 291,1304-1351) and these members fall into three classes (class I, II, andIV) based on sequence homology to their yeast orthologues (O. Witt etal. Cancer Letters, 2009, 277, 8-21). Class I HDACs include HDAC1,HDAC2, HDAC3, and HDAC8, and are referred to as “classical” HDACs which,implies a catalytic pocket with a Zn²⁺ ion at its base.

There remains a need for preparing structurally diverse HDAC inhibitors,particularly ones that are potent and/or selective inhibitors ofparticular classes of HDACs and individual HDACs.

SUMMARY OF THE INVENTION

Provided herein are compounds, pharmaceutical compositions comprisingsuch compounds, and methods of using such compounds and compositions totreat or prevent diseases or disorders associated with HDAC activity,particularly diseases or disorders that involve any form of HDAC3expression. Such diseases include hemoglobinopathy, sickle cell disease(SCD), thalassemia, hematologic malignancies, solid tumors andneurodegenerative diseases.

Thus, in one aspect, provided herein is a compound of Formula I:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a compound of Formula II:

or a pharmaceutically acceptable salt thereof.

In yet another aspect, provided herein is a compound of Formula III:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a compound of Formula IV:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a pharmaceutical compositioncomprising any of the compounds of the instant invention (Formula I, II,III, IV, or any of the compounds presented in Table 1) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier.

In another aspect, provided herein is a method of inhibiting HDAC3 in asubject, comprising administering a compound of Formula I, II, III, IV,or any of the compounds presented in Table 1, or a pharmaceuticallyacceptable salt thereof.

In another aspect, provided herein is a method of selectively inhibitingHDAC3 over other HDACs in a subject, comprising administering a compoundof Formula I, II, III, IV, or any of the compounds presented in Table 1,or a pharmaceutically acceptable salt thereof. In one embodiment, thecompound has a selectivity of 5 to 1000 fold for HDAC3. In anotherembodiment, the compound has a selectivity for HDAC3 when tested in aHDAC enzyme assay of about 5 to 1000 fold.

In another aspect, provided herein is a method of treating a diseasemediated by HDAC3 in a subject comprising administering to the subject acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1, or a pharmaceutical composition thereof.

In yet another aspect, provided herein is a method of treating a diseasein a subject comprising administering to the subject a compound ofFormula I, II, III, IV, or any of the compounds presented in Table 1, ora pharmaceutical composition thereof.

In one embodiment, provided herein is a method of treatinghemoglobinopathy in a subject comprising administering to the subject acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1, or a pharmaceutical composition thereof. In another embodiment,provided herein is a method of treating sickle cell disease (SCD) in asubject comprising administering to the subject a compound of Formula I,II, III, IV, or any of the compounds presented in Table 1, or apharmaceutical composition thereof. In another embodiment, providedherein is a method of treating thalassemia in a subject comprisingadministering to the subject a compound of Formula I, II, III, IV, orany of the compounds presented in Table 1, or a pharmaceuticalcomposition thereof.

In certain embodiments, provided herein is a method of treating a canceror a proliferation disease in a subject comprising administering to thesubject a compound of Formula I, II, III, IV, or any of the compoundspresented in Table 1, or a pharmaceutical composition thereof. In oneembodiment, the cancer is a hematologic malignancy. The hematologicmalignancy can be leukemia or lymphoma. The leukemia can further beacute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),acute monocytic leukemia (AML), or acute promyelocytic leukemia (APL).The lymphoma can be Hodgkin's lymphoma or Non-Hodgkin's lymphoma. In yetanother embodiment, the cancer is a solid tumor. The solid tumor can bea brain tumor, neuroblastoma, retinoblastoma, Wilm's tumor, bone tumors,soft-tissue sarcomas, head cancer, neck cancer, gastric cancer, prostatecancer, bladder cancer, renal cancer, cancer of the uterus, ovariancancer, testicular cancer, colon cancer, lung cancer, or breast cancer.The solid tumor can also be gastric cancer, prostate cancer, or coloncancer.

In other embodiments, provided herein is a method of treating aneurodegenerative disease in a subject comprising administering to thesubject a compound of Formula I, II, III, IV, or any of the compoundspresented in Table 1, or a pharmaceutical composition thereof. Theneurodegenerative disease can be Huntington's disease, Friedrich'sataxia, myotonic dystrophy, Parkinson's disease, spinocerebellar ataxia,Kennedy's disease, amyotrophic lateral sclerosis, spinal and bulbarmuscular atrophy, or Alzheimer's disease.

In certain embodiment, provided herein is a method of treating a diseasewhich causes memory deficits comprising administering to the subject acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1, or a pharmaceutical composition thereof.

In other embodiments, provided herein is a method of treating metabolicdisease in a subject comprising administering to the subject a compoundof Formula I, II, III, IV, or any of the compounds presented in Table 1,or a pharmaceutical composition thereof. The metabolic disease can betype 2 diabetes.

In a further embodiment of the methods of treatment described herein,the subject is a human.

DETAILED DESCRIPTION OF THE INVENTION

HDAC inhibitors are capable of de-repressing γ-globin gene expressionand inducing production of HbF (α₂γ₂). Clinical severity ofhemoglobinopathy and thalassemia can be reduced by enhancing expressionof fetal hemoglobin (γ-globin), producing HbF (α₂γ₂) (Boosalis et al.,Blood Cells, Molecules, and Diseases 2011, 15, 107). Sickle cell disease(SCD) is also demonstrated to be treated by induction of fetalhemoglobin (HbF) (Mabaera et al., Experimental Hematology 2008, 1057).Therefore, HDAC inhibitors are promising molecules for treatment ofhemoglobinopathy, thalassemia and SCD.

In addition, HDACs play an important role in cell proliferation anddifferentiation. HDAC inhibition has been demonstrated modulating thebalance between pro- and anti-apoptotic proteins, upregulating theintrinsic and extrinsic apoptosis pathway, and leading to cell cyclearrest. Thus, HDACs are a compelling therapeutic target for cancertherapy. Some HDAC inhibitors have been shown to induce differentiationand cell death in myeloid and lymphoid model systems (Melnick et al.,Current Opinion in Hematology 2002, 9, 322). In particular, HDAC3 isidentified as a key component of the aberrant transcription regulationin acute promyelocytic leukemia cells (Witt et al., Cancer Letters 2009,277, 8). Therefore, HDAC inhibitors, and HDAC3 inhibitors in particular,can provide benefit to treatment of hematologic malignancies.

HDAC3 is also found to be significantly associated with poor prognosisin large series of gastric, prostate and colorectal cancers (Witt etal., Cancer Letters 2009, 277, 8). Another study of a variety of humancancers indicates that HDAC3 may be one of the most frequentlyupregulated genes in cancer cells (Spurling et al., MolecularCarcinogenesis 2008, 47, 137). Together with HDAC1 and HDAC2, HDAC3 isfound overexpressed in colonic tumors and thus may be an importanttarget for cancer therapy (Fakih et al., Clinical Cancer Research 2010,16, 3786). In addition, cell lines from other cancers, such as breastcancer, hepatic cancer and neuronal cancer, are found to be induced todifferentiate by HDAC inhibitors.

Furthermore, HDAC inhibitors have been found to be protective indifferent cellular and animal models of acute and chronicneurodegenerative diseases, such as Huntington's disease, as well asspinal and bulbar muscular atrophy (Kozikowski et al., J. Med. Chem.2007, 50, 3054). Some studies suggest that HDAC inhibitors can reducememory deficits and neurodegeneration in animal models of Alzheimerdisease and improve learning behavior (Beglopoulos et al., Trends inPharmacological Sciences 2006, 27, 33; Fischer et al., Nature 2007, 447,178). A recent study found that selective inhibition of HDAC3significantly enhanced long-term memory in a persistent manner (McQuownet al., J Neurosci 2011, 31, 764). However, the mechanism of thereported improvement of neurodegenerative diseases and long-term memoryformation by HDAC inhibitors is not fully understood. Thus, inhibitionof HDAC3 with the compounds disclosed herein can be advantageous in thetreatment of neurodegenerative diseases and the improvement of long-termmemory formation.

Class I HDAC inhibitors have also been demonstrated to reduce bodyweight and glucose and insulin levels in obese diabetic mice (Galmozziet al., Diabetes 2012). Thus, class I HDAC inhibitors have beensuggested to be potential therapeutics for type 2 diabetes (Mihaylova etal., Cell 2011, 145, 607; Galmozzi et al., Diabetes 2012). TargetingHDAC1 and HDAC3 is found to be able to provide optimal protection ofbeta cell mass and function in clinical islet transplantation andrecent-onset type 1 diabetic patients (Lundh et al., Diabetologia 2012,55, 2421).

Accordingly, provided herein are compounds, pharmaceutical compositionscomprising such compounds, and methods of using such compounds andcompositions to treat or prevent diseases or disorders associated withHDAC activity. The compounds are, in particular, able to inhibit HDAC3activity.

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “alkyl,” as used herein, refers to saturated, straight- orbranched-chain hydrocarbon moieties containing, in certain embodiments,between one and six, or one and eight carbon atoms, respectively.Examples of C₁-C₆ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl,n-hexyl moieties; and examples of C₁-C₈ alkyl moieties include, but arenot limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl, n-hexyl, heptyl, and octyl moieties.

The term “alkenyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbondouble bond. The double bond may or may not be the point of attachmentto another group. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl,octenyl and the like.

The term “alkynyl,” as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety containing, in certain embodiments, from twoto six, or two to eight carbon atoms having at least one carbon-carbontriple bond. The alkynyl group may or may not be the point of attachmentto another group. Representative alkynyl groups include, but are notlimited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl,octynyl and the like.

The term “aryl,” as used herein, refers to a mono- or poly-cycliccarbocyclic ring system having one or more aromatic rings, fused ornon-fused, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like.

The term “cycloalkyl,” as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated or partially unsaturedcarbocyclic ring compound. Examples of C₃-C₈-cycloalkyl include, but notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclopentyl and cyclooctyl; and examples of C₃-C₁₂-cycloalkyl include,but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Also contemplated aremonovalent groups derived from a monocyclic or polycyclic carbocyclicring compound having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. Examples of such groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term “heteroaryl,” as used herein, refers to a mono- or poly-cyclic(e.g., bi-, or tri-cyclic or more) fused or non-fused, moieties or ringsystem having at least one aromatic ring, having from five to ten ringatoms of which one ring atom is selected from S, O and N; zero, one ortwo ring atoms are additional heteroatoms independently selected from S,O and N; and the remaining ring atoms are carbon. Heteroaryl includes,but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heterocycloalkyl,” as used herein, refers to a non-aromatic3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused ofnon-fused system, where (i) each ring contains between one and threeheteroatoms independently selected from oxygen, sulfur and nitrogen,(ii) each 5-membered ring has 0 to 1 double bonds and each 6-memberedring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatomsmay optionally be oxidized, (iv) the nitrogen heteroatom may optionallybe quaternized, and (iv) any of the above rings may be fused to abenzene ring. Representative heterocycloalkyl groups include, but arenot limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, and tetrahydrofuryl.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The terms “optionally substituted” and any other optionallysubstituted group as used herein, refer to groups that are substitutedor unsubstituted by independent replacement of one, two, or three ormore of the hydrogen atoms thereon with substituents including, but notlimited to:

alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl,

—F, —Cl, —Br, —I,

—OH,

—NO₂, —CN,

—NH₂, —NH—C₁-C₁₂-alkyl, —NH-aryl, -dialkylamino,

—O—C₁-C₁₂-alkyl, —O-aryl,

—C(O)—, —C(O)O—, —C(O)NH—, —OC(O)—, —OC(O)O—, —OC(O)NH—, —NHC(O)—,—NHC(O)O—,

—C(O)—C₁-C₁₂-alkyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl,

—C(O)O—C₁-C₁₂-alkyl, —C(O)O—C₃-C₁₂-cycloalkyl, —C(O)O-aryl,—C(O)O-heteroaryl, —C(O)O-heterocycloalkyl,

—CONH₂, —CONH—C₁-C₁₂-alkyl, —CONH-aryl,

—OCO₂—C₁-C₁₂-alkyl, —OCO₂-aryl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl, —OCONH—aryl,

—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)-aryl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂— aryl,

—S(O)—C₁-C₁₂-alkyl, —S(O)-aryl, —SO₂NH—C₁-C₁₂-alkyl, —SO₂NH— aryl,

—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂-aryl,

—SH, —S—C₁-C₁₂-alkyl, or —S-aryl.

In certain embodiments, the optionally substituted groups include thefollowing: C₁-C₁₂-alkyl, C₂-C₁₂-alkenyl, C₂-C₁₂-alkynyl,C₃-C₁₂-cycloalkyl, C₃-C₁₂-aryl, C₃-C₁₂-heterocycloalkyl, orC₃-C₁₂-heteroaryl.

It is understood that the aryls, heteroaryls, alkyls, and the like canbe further substituted.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable include,but are not limited to, nontoxic acid addition salts are salts of anamino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

This invention also encompasses pharmaceutical compositions containing,and methods of treating disorders through administering,pharmaceutically acceptable prodrugs of compounds of the invention. Forexample, compounds of the invention having free amino, amido, hydroxy orcarboxylic groups can be converted into prodrugs. Prodrugs includecompounds wherein an amino acid residue, or a polypeptide chain of twoor more (e.g., two, three or four) amino acid residues is covalentlyjoined through an amide or ester bond to a free amino, hydroxy orcarboxylic acid group of compounds of the invention. The amino acidresidues include but are not limited to the 20 naturally occurring aminoacids commonly designated by three letter symbols and also includes4-hydroxyproline, hydroxyysine, demosine, isodemosine,3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid,citrulline, homocysteine, homoserine, ornithine and methionine sulfone.Additional types of prodrugs are also encompassed. For instance, freecarboxyl groups can be derivatized as amides or alkyl esters. Freehydroxy groups may be derivatized using groups including but not limitedto hemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug DeliveryReviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groupsare also included, as are carbonate prodrugs, sulfonate esters andsulfate esters of hydroxy groups. Derivatization of hydroxy groups as(acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may bean alkyl ester, optionally substituted with groups including but notlimited to ether, amine and carboxylic acid functionalities, or wherethe acyl group is an amino acid ester as described above, are alsoencompassed. Prodrugs of this type are described in J. Med. Chem. 1996,39, 10. Free amines can also be derivatized as amides, sulfonamides orphosphonamides. All of these prodrug moieties may incorporate groupsincluding but not limited to ether, amine and carboxylic acidfunctionalities

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. Particularly, in embodiments thecompound is at least 85% pure, more preferably at least 90% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

Compounds of the Invention

In one aspect, the invention provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein

A is bicyclic heteroaryl or bicyclic heterocycloalkyl;

R¹ and R² are each independently selected from H or halo;

R³ is H, heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl wherein theheterocycloalkyl or C₁₋₆-alkyl-heterocycloalkyl groups are optionallysubstituted;

R⁴ and R⁵ are each independently selected from H, C₁₋₆-alkyl,C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₃₋₆-cycloalkyl, C₁₋₆-alkyl-C₃₋₆-cycloalkyl,heterocycloalkyl, C₁₋₆-alkyl-heterocycloalkyl, NR⁶R⁷, O—C₁₋₆-alkyl-OR⁸,C₁₋₆-alkyl-OR⁸, aryl, C₁₋₆-alkyl-aryl, heteroaryl,C₁₋₆-alkyl-heteroaryl, C(O)N(R⁶)-heteroaryl, C(O)N(R⁶)-heterocycloalkyl,C(O)N(R⁶)-aryl, C(O)—NR⁶R⁷, C(O)-heteroaryl, C(O)-heterocycloalkyl,C(O)-aryl, C(O)—C₁₋₆-alkyl, CO₂-heteroaryl, CO₂-heterocycloalkyl,CO₂-aryl, CO₂—C₁₋₆-alkyl, or C(O)—C₁₋₆-alkyl-heterocycloalkyl, whereinthe cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups areoptionally substituted;

R⁶ and R⁷ are each independently selected from H, C₁₋₆-alkyl,C₁₋₆-alkyl-OR⁸, CO₂R⁸, or C₁-C₆-alkyl-aryl; and

R⁸ is H or C₁₋₆-alkyl.

In one embodiment of the compound of Formula I, the heterocycloalkyl orC₁₋₆-alkyl-heterocycloalkyl groups are optionally substituted withC₁₋₄-alkyl;

and the cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups of R⁴and R⁵ are optionally substituted with C₁₋₄-alkyl, CO₂R⁸, C(O)R⁸, orC₁₋₆-alkyl-OR⁸.

In another embodiment of the compound of Formula I, A is a bicyclicheteroaryl or bicyclic heterocycloalkyl selected from the groupconsisting of

In one embodiment of the compound of Formula I, R¹ is halo. R¹ can befluoro.

In another embodiment of the compound of Formula I, R² is halo. R² canbe chloro.

In another embodiment of the compound of Formula I, R³ isheterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl, and theheterocycloalkyl or C₁₋₆-alkyl-heterocycloalkyl groups can be optionallysubstituted with C₁₋₄-alkyl. R³ can be piperazinyl or piperazinyl-CH₃.R³ can also be CH₂CH₂-morpholinyl.

In another embodiment of the compound of Formula I, R⁴ is independentlyselected from H, C₃₋₆-cycloalkyl, C₁₋₆-alkyl-C₃₋₆-cycloalkyl,heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl. R⁴ can also beindependently selected from H or C₃₋₆-cycloalkyl. R⁴ can be H only. R⁴can also be C₃₋₆-cycloalkyl only.

In yet another embodiment of the compound of Formula I, R⁵ is H.

In another aspect, the invention provides a compound of Formula II:

or a pharmaceutically acceptable salt thereof,

wherein

A is bicyclic heteroaryl or bicyclic heterocycloalkyl;

R¹ and R² are each independently selected from H or halo;

R³ is H, heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl wherein theheterocycloalkyl or C₁₋₆-alkyl-heterocycloalkyl groups are optionallysubstituted;

R⁴ and R⁵ are each independently selected from H, C₁₋₆-alkyl,C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₃₋₆-cycloalkyl, C₁₋₆-alkyl-C₃₋₆-cycloalkyl,heterocycloalkyl, C₁₋₆-alkyl-heterocycloalkyl, NR⁶R⁷, O—C₁₋₆-alkyl-OR⁸,C₁₋₆-alkyl-OR⁸, aryl, C₁₋₆-alkyl-aryl, heteroaryl,C₁₋₆-alkyl-heteroaryl, C(O)N(R⁶)-heteroaryl, C(O)N(R⁶)-heterocycloalkyl,C(O)N(R⁶)-aryl, C(O)—NR⁶R⁷, C(O)-heteroaryl, C(O)-heterocycloalkyl,C(O)-aryl, C(O)—C₁₋₆-alkyl, CO₂-heteroaryl, CO₂-heterocycloalkyl,CO₂-aryl, CO₂—C₁₋₆-alkyl, or C(O)—C₁₋₆-alkyl-heterocycloalkyl, whereinthe cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups areoptionally substituted;

R⁶ and R⁷ are each independently selected from H, C₁₋₆-alkyl,C₁₋₆-alkyl-OR⁸, CO₂R⁸, or C₁-C₆-alkyl-aryl; and

R⁸ is H or C₁₋₆-alkyl.

In one embodiment of the compound of Formula II, R¹ is fluoro.

In another embodiment of the compound of Formula II, R³heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl, and theheterocycloalkyl or C₁₋₆-alkyl-heterocycloalkyl groups can be optionallysubstituted. R³ can be piperazinyl or piperazinyl-CH₃. R³ can also beCH₂CH₂-morpholinyl.

In another embodiment of the compound of Formula II, R⁴ is independentlyselected from H, C₃₋₆-cycloalkyl, C₁₋₆-alkyl-C₃₋₆-cycloalkyl,heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl. R⁴ can also beindependently selected from H or C₃₋₆-cycloalkyl. R⁴ can be H only. R⁴can also be C₃₋₆-cycloalkyl only.

In yet another embodiment of the compound of Formula II, R⁵ is H.

In another aspect, the invention provides a compound of Formula III:

or a pharmaceutically acceptable salt thereof,

wherein

A is bicyclic heteroaryl or bicyclic heterocycloalkyl;

R¹ and R² are halo;

R³ is H, heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl;

R⁴ and R⁵ are each independently selected from H, C₁₋₆-alkyl,C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₃₋₆-cycloalkyl, C₁₋₆-alkyl-C₃₋₆-cycloalkyl,heterocycloalkyl, C₁₋₆-alkyl-heterocycloalkyl, NR⁶R⁷, O—C₁₋₆-alkyl-OR⁸,C₁₋₆-alkyl-OR⁸, aryl, C₁₋₆-alkyl-aryl, heteroaryl,C₁₋₆-alkyl-heteroaryl, C(O)N(R⁶)-heteroaryl, C(O)N(R⁶)-heterocycloalkyl,C(O)N(R⁶)-aryl, C(O)—NR⁶R⁷, C(O)-heteroaryl, C(O)-heterocycloalkyl,C(O)-aryl, C(O)—C₁₋₆-alkyl, CO₂-heteroaryl, CO₂-heterocycloalkyl,CO₂-aryl CO₂—C₁₋₆-alkyl, or C(O)—C₁₋₆-alkyl-heterocycloalkyl, whereinthe cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups areoptionally substituted;

R⁶ and R⁷ are each independently selected from H, C₁₋₆-alkyl,C₁₋₆-alkyl-OR⁸, CO₂R⁸, or C₁-C₆-alkyl-aryl; and

R⁸ is H or C₁₋₆-alkyl.

In one embodiment of the compound of Formula III, R¹ is chloro and R² isfluoro.

In another embodiment of the compound of Formula III, R³ is H, R⁴ is H,and R⁵ is H.

In another aspect, the invention provides a compound of Formula IV:

or a pharmaceutically acceptable salt thereof,

wherein

A is bicyclic heteroaryl or bicyclic heterocycloalkyl;

R¹ is H or halo;

R³ is heterocycloalkyl, or C₁₋₆-alkyl-heterocycloalkyl;

R⁴ is selected from C₃₋₆-cycloalkyl, C₁₋₆-alkyl-C₃₋₆-cycloalkyl,heterocycloalkyl, C₁₋₆-alkyl-heterocycloalkyl, aryl, C₁₋₆-alkyl-aryl,heteroaryl, C₁₋₆-alkyl-heteroaryl wherein the cycloalkyl,heterocycloalkyl, aryl, or heteroaryl groups are optionally substituted.

In one embodiment of the compound of Formula IV, R¹ is halo. R¹ can befluoro.

In another embodiment of the compound of Formula IV, R³ isheterocycloalkyl. R³ can be piperazinyl.

In another embodiment of the compound of Formula IV, R⁴ is cycloalkyl.

In yet another embodiment of the compound of Formula IV, R⁴ iscyclopropyl.

In one aspect, provided herein is a compound selected from the groupconsisting of the following compounds in Table 1 below, orpharmaceutically acceptable salts thereof. The assay conditions used toacquire the HDAC inhibition data are described in Example 10.

TABLE 1 Compound IC 50 (nM) ID New Structures HDAC1 HDAC2 HDAC3 A

468 455  59 B

355 372  53 C

No inhibition >2000  269 D

No inhibition >2000  282 E

1606  805  68 F

G

>2000  1532  193 H

>2000  1780  320 I

>2000  589  57 J

No inhibition >2000  470

In preferred embodiments, the compounds of the instant invention haveone or more of the following properties: the compounds are capable ofinhibiting at least one histone deacetylase (HDAC); the compounds arecapable of inhibiting HDAC3; the compounds are selective HDAC3inhibitors.

The invention also provides for a pharmaceutical composition comprisinga compound of instant invention, or a pharmaceutically acceptable saltthereof, together with a pharmaceutically acceptable carrier.

Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) in the manufacture of amedicament for use in the treatment of a disorder or disease herein.Another object of the present invention is the use of a compound or acomposition as described herein (e.g., of any formulae herein) for usein the treatment of a disorder or disease herein.

In another aspect, the invention provides a method of synthesizing acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1. The synthesis of the compounds of the invention can be found inthe examples below.

Another embodiment is a method of making a compound of any of theformulae herein using any one, or combination of, reactions delineatedherein. The method can include the use of one or more intermediates orchemical reagents delineated herein.

Another aspect is an isotopically labeled compound of any of theformulae delineated herein. Such compounds have one or more isotopeatoms which may or may not be radioactive (e.g., ³H, ²H, ¹⁴C, ¹³C, ³⁵S,³²P, ¹²⁵I, and ¹³¹I) introduced into the compound. Such compounds areuseful for drug metabolism studies and diagnostics, as well astherapeutic applications.

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., Bioorganic and Medicinal Chemistry Letters1994, 4, 1985). For example, appropriate prodrugs can be prepared byreacting a non-derivatized compound of the invention with a suitablecarbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3rd edition, John Wiley and Sons, Inc.,1999, and subsequent editions thereof.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxan, tetrahydrofuranor methanol.

Acids and bases useful in the methods herein are known in the art. Acidcatalysts are any acidic chemical, which can be inorganic (e.g.,hydrochloric, sulfuric, nitric acids, aluminum trichloride) or organic(e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid,ytterbium triflate) in nature. Acids are useful in either catalytic orstoichiometric amounts to facilitate chemical reactions. Bases are anybasic chemical, which can be inorganic (e.g., sodium bicarbonate,potassium hydroxide) or organic (e.g., triethylamine, pyridine) innature. Bases are useful in either catalytic or stoichiometric amountsto facilitate chemical reactions.

In addition, some of the compounds of this invention have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. All such isomeric forms of these compoundsare expressly included in the present invention. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Thecompounds of this invention may also be represented in multipletautomeric forms, in such instances, the invention expressly includesall tautomeric forms of the compounds described herein. When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion. All such isomeric forms of suchcompounds are expressly included in the present invention. All crystalforms of the compounds described herein are expressly included in thepresent invention.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired compounds of the present invention.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein are known in the art and include, for example, thosesuch as described in R. Larock, Comprehensive Organic Transformations,VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995), and subsequenteditions thereof.

The compounds of this invention may be modified by appending variousfunctionalities via any synthetic means delineated herein to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. Where a compound is referred to byboth a chemical structure and a chemical name, and the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the compound's identity.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Methods of the Invention

In one aspect, the invention provides a method of inhibiting HDAC3 in asubject, comprising administering a compound of Formula I, II, III, IV,or any of the compounds presented in Table 1, or a pharmaceuticallyacceptable salt thereof.

In another aspect, the invention provides a method of selectivelyinhibiting HDAC3 over other HDACs in a subject, comprising administeringa compound of Formula I, II, III, IV, or any of the compounds presentedin Table 1, or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound has a selectivity of 5 to 1000 fold forHDAC3. In another embodiment, the compound has a selectivity for HDAC3when tested in a HDAC enzyme assay of about 5 to 1000 fold.

In another aspect, the invention provides a method of treating a diseasemediated by HDAC3 in a subject comprising administering to the subject acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1, or a pharmaceutical composition thereof.

In yet another aspect, the invention provides a method of treating adisease in a subject comprising administering to the subject a compoundof Formula I, II, III, IV, or any of the compounds presented in Table 1,or a pharmaceutical composition thereof.

Inhibition of HDAC3 de-represses γ-globin gene expression and inducesproduction of fetal hemoglobin (HbF). Induction of HbF is an establishedapproach to treat hemoglobinopathy, thalassemia and sickle cell disease(SCD). Thus, the compounds and compositions are capable of de-repressingfetal hemoglobin through HDAC inhibition. In one embodiment, theinvention provides a method of treating hemoglobinopathy in a subjectcomprising administering to the subject a compound of Formula I, II,III, IV, or any of the compounds presented in Table 1, or apharmaceutical composition thereof. In another embodiment, the inventionprovides a method of treating sickle cell disease (SCD) in a subjectcomprising administering to the subject a compound of Formula I, II,III, IV, or any of the compounds presented in Table 1, or apharmaceutical composition thereof. In yet another embodiment, theinvention provides a method of treating thalassemia in a subjectcomprising administering to the subject a compound of Formula I, II,III, IV, or any of the compounds presented in Table 1, or apharmaceutical composition thereof.

HDACs also play an important role in cell proliferation anddifferentiation. Inhibition of HDACs induces apoptosis. Thus HDACinhibitors can be useful in the treatment of cancers, by effecting tumorcell death or inhibiting the growth of tumor cells. In certainembodiments, to the invention provides a method of treating a cancer ora proliferation disease in a subject comprising administering to thesubject a compound of Formula I, II, III, IV, or any of the compoundspresented in Table 1, or a pharmaceutical composition thereof.

In another embodiment, the cancer is a hematologic malignancy. Thehematologic malignancy can be leukemia or lymphoma. The leukemia canfurther be acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenousleukemia (CML), acute monocytic leukemia (AML), or acute promyelocyticleukemia (APL). The lymphoma can be Hodgkin's lymphoma or Non-Hodgkin'slymphoma.

In yet another embodiment, the cancer is a solid tumor. The solid tumorcan be a brain tumor, neuroblastoma, retinoblastoma, Wilm's tumor, bonetumors, soft-tissue sarcomas, head cancer, neck cancer, gastric cancer,prostate cancer, bladder cancer, renal cancer, cancer of the uterus,ovarian cancer, testicular cancer, colon cancer, lung cancer, or breastcancer. The solid tumor can also be gastric cancer, prostate cancer, orcolon cancer.

Furthermore, HDAC inhibitors have been found to be protective indifferent cellular and animal models of acute and chronicneurodegenerative diseases. Thus, in one embodiment, the inventionprovides a method of treating a neurodegenerative disease in a subjectcomprising administering to the subject a compound of Formula I, II,III, IV, or any of the compounds presented in Table 1, or apharmaceutical composition thereof.

In certain embodiments, the neurodegenerative disease is Huntington'sdisease, Friedrich's ataxia, myotonic dystrophy, Parkinson's disease,spinocerebellar ataxia, Kennedy's disease, amyotrophic lateralsclerosis, spinal and bulbar muscular atrophy, or Alzheimer's disease.

HDAC inhibitors have been demonstrated to be able to reduce memorydeficits and neurodegeneration. In particular, inhibition of HDAC3 isfound to significantly enhance long-term memory. Thus, in oneembodiment, the invention provides a method of treating a disease whichcauses memory deficits comprising administering to the subject acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1, or a pharmaceutical composition thereof.

Class I HDAC inhibitors have also been demonstrated to reduce bodyweight and glucose and insulin levels in obese diabetic mice, and aresuggested to be potential therapeutics for type 2 diabetes. Thus, in oneembodiment, the invention provides a method of treating metabolicdisease in a subject comprising administering to the subject a compoundof Formula I, II, III, IV, or any of the compounds presented in Table 1,or a pharmaceutical composition thereof.

In certain embodiment, the invention provides a method of treating type2 diabetes in a subject comprising administering to the subject acompound of Formula I, II, III, IV, or any of the compounds presented inTable 1, or a pharmaceutical composition thereof.

Methods delineated herein include those wherein the subject isidentified as in need of a particular stated treatment. Identifying asubject in need of such treatment can be in the judgment of a subject ora health care professional and can be subjective (e.g. opinion) orobjective (e.g. measurable by a test or diagnostic method).

Also, as discussed above, the compounds of the invention are selectiveinhibitors of HDAC3 and, as such, are useful in the treatment ofdisorders modulated by these histone deacetylases (HDACs). For example,compounds of the invention may be useful in the treatment of cancer(e.g., lung cancer, colon cancer, breast cancer, leukemia, or lymphomas,etc.). Accordingly, in yet another aspect, according to the methods oftreatment of the present invention, tumor cells are killed, or theirgrowth is inhibited by contacting said tumor cells with an inventivecompound or composition, as described herein.

Thus, in another aspect of the invention, methods for the treatment ofcancer are provided comprising administering a therapeutically effectiveamount of an inventive compound (i.e., of any of the formulae herein),as described herein, to a subject in need thereof. In certainembodiments, the subject is identified as in need of such treatment. Incertain embodiments, a method for the treatment of cancer is providedcomprising administering a therapeutically effective amount of aninventive compound, or a pharmaceutical composition comprising aninventive compound to a subject in need thereof, in such amounts and forsuch time as is necessary to achieve the desired result. In certainembodiments of the present invention a “therapeutically effectiveamount” of the inventive compound or pharmaceutical composition is thatamount effective for killing or inhibiting the growth of tumor cells.The compounds and compositions, according to the method of the presentinvention, can be administered using any amount and any route ofadministration effective for killing or inhibiting the growth of tumorcells. Thus, the expression “amount effective to kill or inhibit thegrowth of tumor cells,” as used herein, refers to a sufficient amount ofagent to kill or inhibit the growth of tumor cells. The exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the severity of theinfection, the particular anticancer agent, its mode of administration,and the like.

In certain embodiments, the method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of it. In certain embodiments, theinventive compounds as useful for the treatment of cancer and otherproliferative disorders including, but not limited to lung cancer (e.g.non-small cell lung cancer), colon and rectal cancer, breast cancer,prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, glioma, gliobastoma, hepatocellularcarcinoma, papillary renal carcinoma, head and neck squamous cellcarcinoma, leukemia (e.g., CML, AML, CLL, ALL), lymphomas (non-Hodgkin'sand Hodgkin's), myelomas, retinoblastoma, cervical cancer, melanomaand/or skin cancer, bladder cancer, uterine cancer, testicular cancer,esophageal cancer, and solid tumors.

In certain embodiments, the invention provides a method of treatment ofany of the disorders described herein, wherein the subject is a human.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount of a compound of the invention or a pharmaceutically acceptablesalt thereof. For any of the above uses, the required dosage will varydepending on the mode of administration, the particular condition to betreated and the effect desired.

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising any of the compounds of the instant invention (Formula I, II,III, IV, or any of the compounds presented in Table 1) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable carrier.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Pharmaceutical compositions comprising a compound ofthe present invention in free form or in a pharmaceutically acceptablesalt form in association with at least one pharmaceutically acceptablecarrier or diluent can be manufactured in a conventional manner bymixing, granulating or coating methods. For example, oral compositionscan be tablets or gelatin capsules comprising the active ingredienttogether with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol;for tablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

The present invention encompasses pharmaceutically acceptable topicalformulations of inventive compounds. The term “pharmaceuticallyacceptable topical formulation,” as used herein, means any formulationwhich is pharmaceutically acceptable for intradermal administration of acompound of the invention by application of the formulation to theepidermis. In certain embodiments of the invention, the topicalformulation comprises a carrier system. Pharmaceutically effectivecarriers include, but are not limited to, solvents {e.g., alcohols, polyalcohols, water), creams, lotions, ointments, oils, plasters, liposomes,powders, emulsions, microemulsions, and buffered solutions (e.g.,hypotonic or buffered saline) or any other carrier known in the art fortopically administering pharmaceuticals. A more complete listing ofart-known carriers is provided by reference texts that are standard inthe art, for example, Remington's Pharmaceutical Sciences, 16th Edition,1980 and 17th Edition, 1985, both published by Mack Publishing Company,Easton, Pa., the disclosures of which are incorporated herein byreference in their entireties. In certain other embodiments, the topicalformulations of the invention may comprise excipients. Anypharmaceutically acceptable excipient known in the art may be used toprepare the inventive pharmaceutically acceptable topical formulations.Examples of excipients that can be included in the topical formulationsof the invention include, but are not limited to, preservatives,antioxidants, moisturizers, emollients, buffering agents, solubilizingagents, other penetration agents, skin protectants, surfactants, andpropellants, and/or additional therapeutic agents used in combination tothe inventive compound. Suitable preservatives include, but are notlimited to, alcohols, quaternary amines, organic acids, parabens, andphenols. Suitable antioxidants include, but are not limited to, ascorbicacid and its esters, sodium bisulfite, butylated hydroxytoluene,butylated hydroxyanisole, tocopherols, and chelating agents like EDTAand citric acid. Suitable moisturizers include, but are not limited to,glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol.Suitable buffering agents for use with the invention include, but arenot limited to, citric, hydrochloric, and lactic acid buffers. Suitablesolubilizing agents include, but are not limited to, quaternary ammoniumchlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates.Suitable skin protectants that can be used in the topical formulationsof the invention include, but are not limited to, vitamin E oil,allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

In certain embodiments, the compositions may be in the form ofointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants or patches. In certain exemplary embodiments, formulations ofthe compositions according to the invention are creams, which mayfurther contain saturated or unsaturated fatty acids such as stearicacid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleylalcohols, stearic acid being particularly preferred. Creams of theinvention may also contain a non-ionic surfactant, for example,polyoxy-40-stearate. In certain embodiments, the active component isadmixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are made by dissolving or dispensing thecompound in the proper medium. As discussed above, penetration enhancingagents can also be used to increase the flux of the compound across theskin. The rate can be controlled by either providing a rate controllingmembrane or by dispersing the compound in a polymer matrix or gel.

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

According to the methods of treatment of the present invention,disorders are treated or prevented in a subject, such as a human orother animal, by administering to the subject a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result. The term“therapeutically effective amount” of a compound of the invention, asused herein, means a sufficient amount of the compound so as to decreasethe symptoms of a disorder in a subject. As is well understood in themedical arts a therapeutically effective amount of a compound of thisinvention will be at a reasonable benefit/risk ratio applicable to anymedical treatment.

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight (0.05to 4.5 mg/m²). An indicated daily dosage in the larger mammal, e.g.humans, is in the range from about 0.5 mg to about 100 mg, convenientlyadministered, e.g. in divided doses up to four times a day or in retardform. Suitable unit dosage forms for oral administration comprise fromca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compounds ofthe present invention may range from about 0.1 mg/kg to about 500 mg/kg(about 0.18 mg/m² to about 900 mg/m²), alternatively from about 1 toabout 50 mg/kg (about 1.8 to about 90 mg/m²). In general, treatmentregimens according to the present invention comprise administration to apatient in need of such treatment from about 10 mg to about 1000 mg ofthe compound(s) of this invention per day in single or multiple doses.Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes, oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate, agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The protein kinaseinhibitors or pharmaceutical salts thereof may be formulated intopharmaceutical compositions for administration to animals or humans.These pharmaceutical compositions, which comprise an amount of theprotein inhibitor effective to treat or prevent a proteinkinase-mediated condition and a pharmaceutically acceptable carrier, areanother embodiment of the present invention.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not to limit the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims. Definitions of variablesin the structures in schemes herein are commensurate with those ofcorresponding positions in the formulae delineated herein.

Example 1 Synthesis of Compound G

Experimental Procedure

Step 1: A mixture of compound 1 (280 mg, 1.59 mmol),4-(2-chloroethyl)morpholine (474 mg, 3.18 mol), and KOH (267 mg, 4.77mmol) in DMSO (5 mL) was stirred at 55° C. for 2 h. TLC was used tomonitor the reaction. To the mixture was added water (10 mL), extractedwith EA (10 ml*2), separated, dried, filtered and concentrated to getcompound 2 (300 mg, 65%) as light red solid.

Step 2: A mixture of compound 2 (300 mg, 1.4 mmol) and NaOH (2.076 mmol,83 mg) in EtOH (10 mL) was stirred at 60° C. for 3 h. The mixture wasconcentrated to get a residue, to which was added sat. citric acid (10mL), extracted with EA (25 mL×2), separated organic layer, dried,filtered and concentrated to get compound 3 (210 mg, 74%) as a lightyellow solid.

Step 3: A mixture of compound 3 (210 mg, 0.7 mmol), amine (199 mg, 0.7mmol), HOAT (220 mg, 1.05 mmol), EDCI (269 mg, 1.4 mmol), DIPEA (271 mg,2.1 mmol) in DMF (5 mL) was stirred at 55° C. for overnight. To themixture was added water (10 mL), extracted with EA (15 mL×2), theorganic layer was separated, dried, filtered and concentrated to get aresidue, which was purified by Pre-TLC to afford compound 4 (180 mg,47%) as yellow solid.

Step 4: To a solution of compound 6 (180 mg, 0.37 mmol) in DCM (10 mL)was added TFA (2 mL), the reaction was stirred at rt for 2 h. Themixture was worked up and purified by prep-HPLC to afford compound G aswhite solid (10 mg, 7%) LCMS: m/z=384 (M+H)

Example 2 Synthesis of Compound I

Experimental Procedure

Step 1: A mixture of compound 1 (1.0 g, 0.0026 mol) and NBS (0.7 g,0.0039 mol) in DCM was stirred at rt for 3 h. The mixture wasconcentrated to get a residue which was purified by prep-TLC to get thecompound 2 (1.0 g, 83%) as light yellow solid.

Step 2: A mixture of compound 2 (500 mg, 0.0011 mol), cyclopropylboronic acid (946 mg, 0.011 mol), Pd(OAc)₂ (25 mg, 0.00011 mol),tricyclohexylphosphine (61 mg, 0.00022 mol) and K₃PO₄ (933 mg, 0.0044mol) in toluene (20 mL) and water (3 mL) was stirred at 100° C. under N₂atmosphere overnight. The mixture was cooled, filtered, concentrated toget a residue, which was purified by Pre-TLC to get the compound 3 (370mg, 81%) as light yellow solid.

Step 3: A mixture of compound 3 (300 mg, 0.0007 mol) and NaOH (2M, 5 mL)in EtOH (15 mL) and THF (15 mL) was stirred at 60° C. for 5 h. Themixture was concentrated to get a residue, which was added sat. citricacid (10 mL), extracted with EA (25 mL×2), separated organic layer,dried, filtered and concentrated to get compound 4 (195 mg, 70%) aslight yellow solid.

Step 4: A mixture of compound 4 (120 mg, 0.0003 mol), amine (68 mg,0.0003 mol), HOAT (61 mg, 0.00045 mol), EDCI (115 mg, 0.0006 mol), DIPEA(155 mg, 0.0012 mol) in DMF (5 mL) was stirred at 55° C. for overnight.To the mixture was added water (20 mL), extracted with EA (25 mL×2), theorganic layer was separated, dried, filtered and concentrated to get aresidue, which was purified by Pre-TLC to afford compound 5 (145 mg,80%) as yellow solid.

Step 5: A solution of compound 5 (100 mg, 0.00016 mol) in DCM (3 mL) wasadded TFA 1.5 mL) and stirred at rt for 1 h. The mixture was worked upand concentrated to get a residue, which was purified by Pre-HPLC toafford compound I (40 mg, 60%) as white solid.

Example 3 Synthesis of Compound C

Experimental Procedure

Step 1: A mixture of compound 1 (200 mg, 1.0 mmol) and HCl (2.0 M inH2O, 5 mL) was heated to 100° C. for overnight. The mixture wasconcentrated to get compound 2 (180 mg, 97%) as light yellow solid.

Step 2: A solution of compound 2 (90 mg, 0.5 mmol), EDCI (192 mg, 1.0mmol), HOAT (136 mg, 1.0 mmol) and DIPEA (0.5 mL) in DMF (3 mL) wasstirred at rt for 10 min, amine (115 mg, 0.5 mmol) was added, then themixture was heated to 60° C. for overnight. The reaction was quenchedwith water and the precipitate was collected to afford compound 3 (100mg, 41%) as brown solid

Step 3: To a solution of compound 3 (100 mg, 0.25 mmol) in DCM (3 mL)was added TFA (0.6 mL) at 0° C., and then the reaction solution wasstirred at rt for 45 mins. The mixture was concentrated to get aresidue, which was purified by prep-HPLC to afford compound C (5 mg, 7%)as white solid.

Example 4 Synthesis of Compound A

Experimental Procedure

Step 1: To a solution of compound 1 (20 g, 0.087 mol) and SM-1 (33 g,0.17 mol) in EtOH (500 mL) was added SnCl₂.2H₂O (87 g, 0.39 mol), themixture was stirred at 90° C. for 12 h. TLC was used to monitor thereaction. The mixture was cooled and evaporated to get the crudeproduct. Added EA (600 mL), H2O (300 mL), Na₂CO₃ (32 g) and stirred for1 h. The organic layer was separated, dried, filtered and concentratedto get compound 2 (15 g, 92%) as yellow solid.

Step 2: A mixture of compound 2 (560 mg, 2 mmol), N-boc-piperazine (1.3g, 7 mmol), Pd₂(dba)₃ (200 mg, 0.2 mmol), Ruphos (200 mg, 0.4 mmol), andCs₂CO₃ (1.4 g, 7.3 mmol) in Toluene (20 mL) was microwaved at (80° C. 10min, 110° C. 30 min, 145° C. 60 min). TLC was used to monitor reaction.To the mixture was added EA (30 mL), and H₂O (30 mL). The organic layerwas separated, washed, dried, filtered and concentrated, petroleum ether(PE) to wash it, get purified compound 3 (620 mg, ˜100%) as yellowsolid.

Step 3: To a solution of compound 3 (2.48 g, 6.44 mmol) in 20 ml DCM wasadded NBS (1.72 g, 9.66 mmol). The mixture was stirred at 0° C. for 2 hTLC monitored reaction completion. Evaporating to get the crude product,then purified by silica gel column to get compound 4 (1.8 g, 63.6%)

Step 4: To a solution of compound 4 (2 g, 4.32 mmol)andcyclopropylboronic acid (928.7 mg, 10.8 mmol) in 20/20 ml toluene/H₂Owas added TCP (123.2 mg, 0.44 mmol), Pd(OAc)₂ (98.6 mg, 0.44 mmol),K₃PO₄ (2.75 g, 13.0 mmol). The mixture was stirred at 100° C. forovernight. LCMS was used to monitor the reaction. Extracted with EA, Theorganic layer was separated, washed, dried, filtered and concentrated,purified by silica gel column to get compound 5 (1 g, 51.8%)

Step 5: A mixture of compound 5 (600 mg, 1.41 mmol) and 2M NaOH (5 ml)in MeOH (10 ml) and THF (10 ml) was stirred 70° C. for 4 h. TLC was usedto monitor the reaction. The mixture was concentrated to a residue.Added water (30 mL) and used citric acid to adjust PHto 4˜5. Extractedwith EA. The organic layer was separated, washed, dried, filtered andconcentrated to get compound 6 (500 mg, 89.2%)

Step 6: To a solution of compound 6 (120 mg, 0.30 mmol), amine (70 mg,0.3 mmol) in 10 ml DMF was added HOAT (81.6 mg, 0.6 mmol), EDCI (114.6mg, 0.6 mmol), DIPEA (193.5 mg, 1.5 mmol). The mixture was stirred at60° C. for overnight LCMS monitored the reaction completion. Added water(10 ml) extracted with EA. The organic layer was separated, washed,dried, filtered and concentrated to get crude product, Purified byprep-TLC to get compound 7 (100 mg, 54.8%)

Step 7: A mixture of compound 7 (100 mg, 0.16 mmol) and 2 ml TFA in 5 mlDCM was stirred rt for 2 h, LCMS was used to monitor the reaction.Evaporated to get crude product, and purified by prep-HPLC to getcompound A (20 mg, 30.8%).

Example 5 Synthesis of Compound J

Experimental Procedure

Step 1: A mixture of compound 3 (600 mg, 1.56 mol) and 2M NaOH (5 ml) inMeOH (10 ml) and THF (10 ml) was stirred 70° C. for 4 h. TLC was used tomonitor the reaction. The mixture was concentrated to a residue, addedwater (30 mL) and used citric acid to adjust PH to 4˜5. Extracted withEA. The organic layer was separated, washed, dried, filtered andconcentrated to get compound 4 (500 mg, 89.2%)

Step 2: To a solution of compound 4 (107 mg, 0.30 mmol) and amine (70mg, 0.3 mmol) in 10 ml DMF was added HOAT (81.6 mg, 0.6 mmol), EDCI(114.6 mg, 0.6 mmol), and DIPEA (193.5 mg, 1.5 mmol). The mixture wasstirred at 60° C. for overnight. LCMS was used to monitor the reaction.Added water (10 ml), and extracted with EA. The organic layer wasseparated, washed, dried, filtered and concentrated to get crude productPurified by prep-TLC to get compound 5 (95 mg, 56%)

Step 3: A mixture of compound 5 (95 mg, 0.16 mmol) and 2 ml TFA in 5 mlDCM was stirred at rt for 2 h. LCMS was used to monitor the reaction.Evaporated to get crude product and purified by prep-HPLC to getcompound J (20 mg, 34.2%).

Example 6 Synthesis of Compound H

Experimental Procedure

Step 1: To a solution of compound 1 (591 mg, 3.36 mol) and SM-1 (1.0 g,6.72 mmol) in DMSO (10 mL) was added KOH (376 mg, 6.72 mmol). Themixture was stirred at rt. for 24 h. TLC was used to monitor thereaction. Added water (30 ml) and extracted with EA. The organic layerwas separated, washed, dried, filtered and concentrated to get crudeproduct. Purified by silica gel column to get compound 2 (400 mg, 70%)

Step 2: A mixture of compound 2 (400 mg, 1.38 mmol) and 15 ml 2 M HClwas stirred at 100° C. for overnight. LCMS was used to monitor thereaction. Concentrated to get crude product 3 (350 mg, 92.2%)

Step 3: To a solution of compound 3 (82.5 mg, 0.3 mmol), and amine (70mg, 0.3 mmol) in 10 ml DMF was added HOAT (81.6 mg, 0.6 mmol), EDCI(114.6 mg, 0.6 mmol), DIPEA (193.5 mg, 1.5 mmol). The mixture wasstirred at 60° C. for overnight. LCMS was used to monitor the reaction.Added water (10 ml) and extracted with EA. The organic layer wasseparated, washed, dried, filtered and concentrated to get crude productPurified by prep-TLC to get compound 4 (70 mg, 48%)

Step 4: A mixture of compound 4 (70 mg, 0.15 mmol) and 2 ml TFA in 5 mlDCM was stirred r.t for 2 h. LCMS was used to monitor the reaction.Evaporated to get crude product and purified by prep-HPLC to getcompound H (20 mg, 36.2%)

Example 7 Synthesis of Compound B

Experimental Procedure

Step 1: To a solution of compound 6 (90 mg, 0.23 mmol),benzene-1,2-diamine (97.9 mg, 0.91 mmol) in 10 ml DMF was added HOAT(61.7 mg, 0.45 mmol), EDCI (86.6 mg, 0.45 mmol), DIPEA (146.2 mg, 1.13mmol). The mixture was stirred at 60° C. for overnight. LCMS was used tomonitor the reaction. Added water (10 ml) and extracted with EA. Theorganic layer was separated, washed, dried, filtered and concentrated toget crude product and purified by prep-TLC to get compound 7 (90 mg,87%)

Step 2: A mixture of compound 7 (90 mg, 0.18 mmol) and 2 ml TFA in 5 mlDCM was stirred r.t for 2 h. LCMS was used to monitor the reaction.Evaporated to get the crude residue and purified it by prep-HPLC to getcompound B (15 mg, 24%).

Example 8 Synthesis of Compound E

Experimental Procedure

Step 1: To a solution of compound 6 (120 mg, 0.30 mmol), amine (70 mg,0.3 mmol) in 10 ml DMF was added HOAT (81.6 mg, 0.6 mmol), EDCI (114.6mg, 0.6 mmol), DIPEA (193.5 mg, 1.5 mmol). The mixture was stirred at60° C. for overnight. LCMS was used to monitor the reaction. Added water(10 ml) and extracted with EA. The organic layer was separated, washed,dried, filtered and concentrated to get a residue and purified it byprep-TLC to get compound 7 (100 mg, 54.8%)

Step 2: A mixture of compound 7 (100 mg, 0.16 mmol) and 2 ml TFA in 5 mlDCM was stirred r.t for 2 h. LCMS was used to monitor the reaction.Concentrated to get compound 8 (67 mg, 100%)

Step 3: To a solution of compound 8 (30 mg, 0.08 mmol), and CH₃I (12.8mg, 0.09 mmol) in 5 ml THF was added Et₃N (32.3 mg, 0.32 mmol). Themixture was stirred at r.t for 2 h. LCMS was used to monitor thereaction. Added 10 ml H₂O and extracted with EA. The organic layer wasseparated, washed, dried, filtered and concentrated, then purified byprep-HPLC get compound E (10 mg, 33.3%).

Example 9 Synthesis of Compound D

Experimental Procedure

Step 1: A mixture of compound 1 (520 mg, 3 mol) and 2M NaOH (5 ml) inMeOH (10 ml) and THF (10 ml) was stirred 70° C. for overnight. TLC wasused to monitor the reaction. The mixture was then concentrated to aresidue, added water (30 mL) and used citric acid to adjust PH to 5˜6.Extracted with EA, the organic layer was separated, washed, dried,filtered and concentrated to get compound 4 as yellow solid (320 mg,67%)

Step 2: To a solution of compound 2 (81 mg, 0.5 mmol), and amine (113mg, 0.5 mmol) in 5 ml DMF was added HOAT (136 mg, 1 mmol), EDCI (191 mg,1 mmol), DIPEA (260 mg, 2 mmol). The mixture was stirred at 60° C. forovernight. LCMS was used to monitor the reaction. Added water (10 ml)and extracted with EA. The organic layer was separated, washed, dried,filtered and concentrated to get crude product 3 as yellow oil (100 mg,crude)

Step 3: A mixture of compound 3 (100 mg, 00.27 mmol) and 2 ml TFA in 5ml DCM was stirred r.t for 2 h. LCMS was used to monitor the reaction.Concentrated to get compound D which was purified by prep-HPLC (20 mg,30%).

Example 10 HDAC Enzyme Assays

Compounds for testing were diluted in DMSO to 50 fold the finalconcentration and a ten point three fold dilution series was made. Thecompounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl,0.001% Tween-20, 0.05% BSA, 20 μM tris(2-carboxyethyl)phosphine) to 6fold their final concentration. The HDAC enzymes (purchased from BPSBiosciences) were diluted to 1.5 fold their final concentration in assaybuffer and pre-incubated with the compounds for 24 hours prior toaddition of the substrate.

The substrate tripeptide substrate 3 (synthesized in house) for eachenzyme was equal to the Km as determined by a substrate titration curve.The enzyme and substrate concentrations used are given in Table 2. Thesubstrates were diluted in assay buffer at 6× their final concentrationwith 0.3 μM sequencing grade trypsin (Sigma). The substrate/trypsin mixwas added to the enzyme/compound mix, the plate was shaken for 60seconds and placed into a Spectramax M5 microtiter plate reader. Thedevelopment of fluorescence was monitored for 30 min and the linear rateof the reaction was calculated. The IC₅₀ was determined using Graph PadPrism by a four parameter curve fit. The IC₅₀ values obtained for thecompounds of this invention are found in Table 1.

TABLE 2 Enzyme concentration Substrate concentration HDAC1 3.5 ng/μl 3.8μM HDAC2 0.2 ng/μl 2.3 μM HDAC3 0.08 ng/μl  3.9 μM

Example 11 HDAC Inhibition Assays

Tissue culture media were first removed from T75 flasks containingconfluent A549 and NCI-H520 lung cells. The flasks were then washed withPBS. 10 ml enzyme free cell dissociation media was added in to theflasks followed by 30-minute incubation at 37 C in 5% CO2. The flaskswere lightly tapped to ensure that any cells still adhering to the flaskwere dislodged before the media with cells were transferred to 50 mltubes.

The media with cells were then spanned and aspirated before 10 mlcomplete growth media were added. Single cell suspensions were thencreated by repeat pipetting. Cells were counted and volume was adjustedto 10^5 cells/ml. 5 ul of cells were added to 384 well plates pre-platedwith 15 ul complete growth media. Testing compounds were dispensed atthe indicated dose ranges. The plates were spanned for 10 seconds at1000 rpm to settle the cells followed by 48 hour incubation at 37 C in5% CO2. 5 ul of MTS reagent were added to the plates followed by 1 hourincubation at 37 C in 5% CO2.

Optical densities were read at 650 nm and 490 nm. Corrected OD values(OD490-OD650) were plotted as displayed. Cisplatin IC50 values werecalculated by Graphpad Prism software and plotted as displayed.

Incorporation by Reference

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended with be encompassed by the following claims.

We claim:
 1. A compound selected from the following:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, together with a pharmaceutically acceptablecarrier.
 3. A method of treating type 2 diabetes, Huntington's disease,spinal and bulbar muscular atrophy, or lung cancer in a subjectcomprising administering to the subject a compound of claim 1 or apharmaceutically acceptable salt thereof.
 4. The method of claim 3,wherein the disease is type 2 diabetes.
 5. The method of claim 3,wherein the disease is Huntington's disease.
 6. The method of claim 3,wherein the disease is spinal and bulbar muscular atrophy.
 7. The methodof claim 3 wherein the disease is lung cancer.
 8. The method of claim 3,wherein the subject is human.
 9. A compound which is:

or a pharmaceutically acceptable salt thereof.
 10. A compound which is:

or a pharmaceutically acceptable salt thereof.
 11. A compound which is:

or a pharmaceutically acceptable salt thereof.